JPH06507303A - Composition used as a therapeutic agent for chronic viral liver disease - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Composition used as a therapeutic agent for chronic viral liver disease The present invention provides a composition for treating chronic viral liver disease. prepared by recombinant DNA technology to provide a therapeutic agent for sexually transmitted liver disease A composition comprising a polypeptide sequence and a carrier. The present invention provides the A DNA sequence encoding the peptide sequence and a transcript for the expression of said DNA sequence. Regarding infected cells.

At least five viruses, namely hepatitis A, B, C, D and E viruses, are rapidly The clinical phase of sexual hepatitis can be initiated. Intestinal hepatitis A and B Hepatitis B and C (formerly called parenteral non-A, non-B hepatitis) are curable. hepatitis B) and hepatitis B can progress to a chronic stage of inflammation, which then develops into liver cirrhosis. It can cause cancer and primary hepatocellular carcinoma.

Hepatitis C and B, their diagnosis and treatment methods, and available information on each virus. There is relatively little data available. Hepatitis D virus is known to be incomplete. It is an RNA virus. Therefore, it is necessary for the helper to develop in the patient. virus and is only found in individuals infected with HBV. Very recently The hepatitis C virus was finally detected, making it easier to diagnose chronic hepatitis C infection. An antibody test (anti-HCV) has been developed. However, there is no cure for this disease. The urgent demand for this is increasing.

Recognition by immunological methods, the causative virus and the life cycle of the virus and D The same is true for chronic hepatitis B, a disease that has been better studied in terms of NA sequences. apply. If the viral DNA persists for more than 10 weeks, or B If hepatitis surface antigen (HBsAg) persists for more than 6 months, the patient has type B He is said to be a chronic carrier of the hepatitis virus.

Approximately 300 million people are thought to have chronic hepatitis B, the majority of which are extremely severe. They are the people who live in the east. Their main risk of infection is from a chronically infected mother Likely during or immediately after birth, as parents transmit the virus to their newborns. . 90% of children infected in this way develop chronic infections during their later lives. It will become a state.

In Western countries, the main Infection occurs by parenteral or sexual transmission.

In the case of type B infection after birth, only 5-10% of infected individuals become chronic carriers. death However, the transmitted virus may be affected by the infection, depending on whether the infected person eliminates the virus or not. It does not cause a clear reaction that indicates whether the substance is retained in the body for life. Therefore, It seems that it is a matter of immunological status, which determines the future physical condition.

HB virus particles (zonal particles) contain different structural proteins, the core protein It consists of protein and surface (S) protein. The latter is the code for the three S-type regions. sequence (ATG), each of which is capable of initiating translation in vivo It is the translation product of the open reading frame that encompasses the whole. These regions are the 5′ 5′ regions of the molecule. They are called brees S1, blees S2 and S in the order from the 3' end. From this ORF Six protein products: major protein glycosyltranslated only from the S region glycosylated form and non-glycosylated form (gp27 and p24) (226 amino acids), one or more or a central protein encoded by BreS2 and S regions with two polysaccharide side chains. (281 amino acids), and finally, by translation of BreSl, BreS2 and S Glycosylated (gp24) and non-glycosylated forms (gp24) of the large protein formed p39) (389-400 amino acids depending on the serotype of the virus) is induced. Ru. The core proteins are HBcAg and HBeAg, the latter protein probably It is probably a processing product of HBcAg.

Zone particles, which are infectious virus particles, contain both core and surface proteins. The filament, on the other hand, consists of a mixture of six surface antigens. S pep When aggregated alone, TiD forms so-called 20 nm particles that are completely non-infectious. do.

Patients infected with the HB virus are considered to have chronic HBV infection. Patients pass through several stages of hepatitis before becoming infected. Immediately after infection, the presence of HBeAg in serum There will be a more characterized infectious period.

Continuing HBs antigenemia despite suppression of HBV replication may result in an abnormality in the patient's cellular genome. Point out the presence of viral DNA sequences integrated within. embedded virus sequence does not allow the host cell to synthesize a complete virus. however , hepatocytes with integrated HBV sequences can only produce HBsAg. This is detectable in the patient's serum and is an indicator of chronic hepatitis B. Sky However, transformed hepatocytes are not lysed by cytotoxic T cells and proliferate, leading to chronic maintenance. It induces either continuous hepatitis (CPH) or chronic active hepatitis (CAM), which leads to This can progress to cirrhosis or primary hepatocellular carcinoma, leading to premature puberty in patients.

Recently, patients with chronic HBV infection have shown a deficiency in endogenous interferon production. It has been established that (Abb et al., 1985: J. Med.

Virol, 16.171-176), HBe in serum (7) Patients with chronic hepatitis B as indicated by the presence of Ag and HBV-DNA (7) This was the rationale for treating patients with interferon alpha (rFNα). . A large number of patient management trials show that administration of interferon alpha is significant compared to control. showed increased hepatitis B virus clearance.

However, patients infected at or near birth may respond to this treatment It seems that there will be no conversion. This phenomenon unfortunately leaves 75% of holders with [ Disable FNα therapy.

Currently, the exact mode of action of interferon alpha on chronic hepatitis B remains unclear. There is even. Its antiviral activity protects infected cells from infection or HBV May reduce viral transcription, translation, and replication in infected cells . Interferon also activates T cells, macrophages and NK cells. and induces the expression of MHC class I proteins, thereby exerting immunoregulatory functions. have

Another approach to treating chronic BW hepatitis is to suppress viral replication and thereby The idea is to provide sufficient protection to enable the host immune system to eliminate the virus. It is. This is an antiviral drug for the treatment of chronically infected individuals with HBV. , for example, testing adenine arabinoside and adenine arabinoside monophosphate. It comes down to that. However, less than half of patients experience persistent or temporary seroconversion. version (from “HBeAg” to “anti-HBe”) to this therapy. responded. A further disadvantage of these antiviral drugs is their immunosuppressive properties.

Other drugs being tested for treatment of chronic carriers include interferon beta, Acycloguanosine (acyclovir), interleukin 2, steroids, e.g. Examples include prednisolone, and combinations thereof. However, some of them None of these treatments were able to provide better results than treatment with interferon alpha. Ta. Only combination therapy involving the administration of IFNα after an initial dose of steroids can increase the response rate in patients.

Based on conventional technology, chimpanzees chronically infected with HBV have been diagnosed with HBsAg (tetanus toxin). This problem cannot be corrected by treatment with anti-HBs or anti-HBs. is known. Furthermore, chronic HBV-infected patients were treated with S-peptide. Attempts have been made to immunize patients. This treatment is effective against anti-HBs in those people. It did not even cause antibody formation.

Moreover, by definition, chronic carriers of hepatitis B virus have HBsA in their serum. is characterized by being detectable. Therefore, the virus S according to the present invention Combinations of peptides containing T-cell activation epitopes have been shown to be effective against chronic hepatitis B virus infection. It was completely unexpected that immunization and eventual healing could be induced in sex carriers.

Considering the state of the art as described above, it is an object of the present invention to achieve a complete response (i.e., HBV Persistent inhibition of replication, loss of HBV DNA and DNA polymerase, and decrease in serum HBeAg and HBsAg and eventually their disappearance). The object of the present invention is to provide an effective therapeutic agent for the treatment of viral chronic liver disease.

According to the invention, this aim is to: a) mediate the antigenicity of one or more epitopes; b) at least one polypeptide sequence representing the epitope sequence a); in combination with a carrier capable of adsorbing, wherein said polypeptide sequence a) combinations that can be bonded to the carrier b) by any chemical bond or subvalence; more achieved.

The invention further provides the use of this combination for the manufacture of a medicament for the treatment of chronic viral hepatitis. Directed.

The invention further provides: a) at least one agent that mediates the antigenicity of one or more epitopes; a carrier capable of presenting one polypeptide sequence and b) an epitope sequence a) , wherein said polypeptide sequence a) is capable of adsorption, any chemical bonding. or administering to the patient said combination which can be attached to the carrier b) by a subvalent. The invention is also directed to a method of treating chronic viral hepatitis.

■or the polypeptide sequence a) can be several different polypeptides; It is important to mediate the antigenicity of T cell activation epitopes by direct or indirect methods. It is essential. According to the invention, polypeptide sequence a) can be of any subtype, in particular One polypep of hepatitis B virus: adw, ayw, adr and ady polypeptides or a combination of two or more polypeptides.

Those peptides derived from hepatitis B virus include HBV peptide pres1, can be S2 or S or HBV core antigen.

Further useful as polypeptide sequences a) are at least 6 contiguous amino acids such that at least one epitope comprising the residue must be conserved. or by a deletion of an amino acid at the N-terminus of the polypeptide sequence a) or at the C-terminus. or modified by adding additional amino acids as insertions into the sequence. , any of the above-mentioned polypeptides or a combination of two or more polypeptides.

However, in each of these cases it is essential that biological activity is retained. be.

Preferably, polypeptide sequence a) is myristylated.

In order to exhibit suitable pharmacological activity, the polypeptide sequence a) is presented on carrier b). This carrier can be made of, for example, a hydrophobic polymer. - consists of certain substances which can consist of particles, inorganic particles or polysaccharide particles. good Preferably, carrier b) is a second polypeptide sequence that forms particles upon secretion. , the particles preferably grow a diameter of at least 10 nm.

The polypeptide sequence b) forming the particles consists of HBV S pebuti, de, HBV core anti- original, HAV core antigen, HAV surface antigen, HIV surface antigen and HIV core antigen and surface antigens of poliovirus. Preferably, it is a substantial portion or a complete amino acid sequence. Particle-forming carrier b) Preferred as HBV S peptide and/or core peptide.

When used as carrier sequence b), the above-mentioned polypeptide may be modified by any amino acid deletions, substitution of one or more amino acids, one or more at either the N-terminus or the C-terminus; is the addition of multiple amino acids or one or more into the polypeptide sequence b) Can be modified by insertion of amino acids, but particle-forming ability is retained. It is assumed that Preferably, polypeptide sequence b) is myristylated .

If carrier b) is a polypeptide sequence, rearrange a) and b) by the following interaction: Hydrophobic anchoring (mediated by myristic acid), disulfide bond formation, among or rearrangement into a peptide bond. can be linked together to form a fusion peptide. In the latter case, if desired Insert a spacer sequence between polypeptide sequence a) and polypeptide sequence b) This spacer sequence connects both polypeptides by a peptide bond. be combined.

The present invention further provides a composition comprising: useful for the manufacture of a medicament for the treatment of chronic viral liver disease; Recombinant DNA molecules encoding combinations are provided. This recombinant DNA molecule at least one first DNA sequence, optionally a second, third and/or fourth D comprises the NA sequence, where i) said at least one first DNA sequence is at least one as defined above; ii) said second DNA sequence encodes a polypeptide sequence of encoding a polypeptide sequence b) according to the above definition of peptide; ii) said third iv) said fourth DNA sequence encodes a spacer sequence; and iv) said fourth DNA sequence encodes a selectable marker, and said DNA sequence is essential for expression and the NA element and optionally have a common reading frame.

Given the fact that many amino acids are specified by multiple triplets, , encoding the peptide sequences a) and b) as defined above, are encompassed by the invention. There are several DNA sequences that exist.

Besides this, the invention relies on the fact that up to 30% of the nucleotides can be substituted. It further encompasses recombinant DNA molecules different from the recombinant DNA molecules defined above. .

A further object of the present application is to provide transfectants with recombinant DNA molecules encoding the above-mentioned combinations. and to provide infected host cells, which host cells are chronically susceptible to HBV infection. It is useful in the treatment of infected patients. This host cell can be a mammalian, yeast or bacterial cell. can be done. For the purposes of the present invention, this host cell is Does not produce any primate or human serum proteins other than peptides. It is preferable that the

As used herein, the term "rHBV S peptide" refers to the entire region of the HBV genome. refers to a peptide that is encoded by The term rHBV used herein 2 peptide” is a peptide encoded by the complete S2 and S regions of the HBV genome. Say petit de. As used herein, the term "rHBV BreSl peptide" refers to H The polypeptide encoded by the complete BreSl, BreS2 and S regions of the BV genome. Say petit de. As used herein, the term "epitope" refers to a specified genomic region. refers to a sequence of at least six contiguous amino acids encoded by (e.g., rH "BV BreS2 epitope" is the epitope encoded by the BreS2 region of the H, BV genome. a sequence of at least six contiguous amino acids). As used herein, the term “ "T cell epitopes" interact with receptors on the surface of T cells to trigger an immune response. Refers to an epitope that enhances or produces an immune response. used in this specification "Antigenicity" refers to the ability to elicit an immune response (e.g. act as an antigen); ability to cause antibody production (e.g. act as an antigen) and/or surface cells. have the ability to interact with cell receptors and enhance immune responses or antibody production. means.

The term "rHBV" is used in the literature (P. Valenzuela, Nature No. 28). Volume 0, page 815 (1979); Gerlich, EP-A-851113 61; Neurath, BP-A-85102250] Any subtype of Rus, especially adw, ayw.

means adr and ayr. mediate the antigenicity of one or more epitopes Examples of the peptide sequences constituting polypeptide sequence a) are shown in the sequence list (SEQ ID NO: 1). 7 to 20.22).

A preferred embodiment of the present invention is a combination of NiHBS antigen particles and BreSl Specific epitopes of the peptide, BreS2 peptide and/or core peptide ( combination with determinant); -HB cocoa raw particles and BreSl peptide, BreS2 peptide, S peptide and / or in combination with specific epitopes (determinants) of the core antigen; - Hepatitis A antigen particles, hepatitis B S peptide, BreSl peptide, BreS2 peptide and/or core peptides with specific epitopes (determinants).

Preferred recombinant DNA molecules for the present invention contain one or more T cell epitopes. a sequence encoding a polypeptide sequence a) that mediates the antigenicity of Sequence encoding polypeptide b) forming particles having a diameter of 0 nm or more characterized by the presence of rearrangements (also under the control of a suitable promoter) . Examples of sequences encoding a) are listed under SEQ ID NOS: 1 to 24 in the sequence listing. Here are some examples of arrays that can be used. DNA sequence encoding polypeptide sequence b) An example of this is represented by any of the sequences SEQ ID NOs: 25 to 27 in the Sequence Listing.

Any of the 24 types of sequences (SEQ ID NOs: 1 to 24) to SEQ ID NOS: 25 to 27 in the sequence list in which a-b and b-a can be combined with the sequences disclosed under Both orders are included.

A particularly preferred embodiment of the invention is SEQ ID NO: 26 and/or The epitope of SEQ ID NO: 28 in combination with sequence 27 (the amino acid of sequence 81 of HBV) (corresponding to 9 to 28 amino acids).

The hepatitis virus sequences used in the recombinant DNA constructs of the invention are restriction fragments. Isolation and ligation, synthesis using a synthesizer (Cycl+) 1.3io-3earch) Chemical synthesis of oligonucleotides and synthesis by PCR method (T, J, White , N., Arnlejm, H. Ei, Erlich, 1989. Th e Polymerase Chain Reaction, Technica 1 Focus 5(6)). I can do it.

A preferred recombinant DNA molecule combines synthetic oligonucleotides into the S region of the HBV genome. 5' XbaI-BglII 3' fragment (SEQ ID NO: 27) from Bgl IF-Bgl IIHBV fragment containing the Bre St-Ble 32-3 region (derived from) or by linking to the complete S region.

Oligonucleotides used to generate such constructs are summarized in Table 1 of the website.

Table■ Other preferred DNA molecules are prepared by PCR methods and contain T cell epitopes. The core sequence encoding the HBV core sequence which functions as the polypeptide sequence b) (SEQ ID NO: 25). for the production of those constructs. The oligonucleotides used are given in Table If-1.

Table 1-2 shows D encoding T cell epitopes by restriction cleavage of the HBV genome. Isolate the NA sequence and prepare a sequence encoding polypeptide sequence b) as defined above. Here are some examples of concatenating columns.

This book sequence 1 Gaibert, F. et al. (1979: Nature 28 1.646-660) and Ono, Y. et al. (1983: Nucl, Ac 1ds Res, 11(6), 1747-1757) .

Table 11-3 lists specific recombinant DNA molecules. The method for producing them is described in the example. It will be described in more detail later.

Table IF-3 ’　egpt=Escherichia coli xanthine guanine phosphoribosyltransferase Preferred recombinant DNA molecules of the invention coat polypeptides a) and b). In addition to the region containing the DNA, it comprises an additional DNA sequence encoding a selectable marker. Change In addition, they contain all the useful elements essential for expression, such as promoter sequences, initiation It comprises codons and polyadenylation sequences.

Examples of suitable promoters include metal promoters when using mammalian cells as host cells. Thionein (MT) is a promoter in U2 and H2. yeast or bacteria If cells are to be used, appropriate yeast and bacterial promoters, such as G CN4 and GAL l/10 promoters, or prokaryotic trp and tac Each promoter can be used.

To produce the combination of polypeptide a) and polypeptide b) according to the invention, , by transfection (in the case of mammalian cells) with recombinant DNA molecules into host cells by transformation (in the case of yeast and bacterial cells) or by other means. Insert into. of any organism capable of transcribing and translating recombinant DNA molecules. Cells such as mammalian, bacterial and yeast cells can be used as hosts .

Suitable mammalian cells according to the invention include, for example, VERO cells (monkey kidney cell line) , 3T3, Cl27 and L cells (mouse fibroblast cell line), and CHO (Chi Chinese hamster ovary) cells, and they contain dehydrofolate reductase. Either positive or negative.

According to a particular embodiment of the invention, the polypeptide sequence a) and the polypeptide sequence b) a first DNA sequence as defined above and a second DNA sequence as defined above encoding It is further possible that the A sequence is present in different recombinant DNA molecules and If the host cell is co-transfected with both of these recombinant DNA molecules, It will be done.

Table 3 shows suitable methods for combining NA sequences to provide the DNA constructs of the present invention. This is a summary of the information. Any of the components shown in this table, in combination, Once transfected into cells, it is taken up and used to treat chronic viral hepatitis. for producing a medicinal combination [comprising polypeptides a) and b)]; It should be noted that it is possible to provide a DNA sequence that can be used. T cell epitome The DNA sequence encoding the loop sequence was obtained using the Biosearch Cyclon synthesis system. synthetically (SYN), by the PCR method (PCR), or with It was prepared by restriction enzyme cleavage of the Rus genome (GENE).

Polypeptide sequence a) and carrier for the treatment of patients with chronic viral hepatitis The combination of b) can be formulated into any type of pharmaceutical composition. The pharmaceutical group The composition further comprises a suitable diluent or pharmaceutical carrier material, such as a buffer.

Administration may be by any method, parenterally (e.g. intravenously or intramuscularly) or orally (e.g. (e.g. by using bacterial cells encapsulating the active substance) .

Pharmaceutical compositions contain the above-mentioned compounds at a concentration sufficient to elicit a response upon administration. It consists of a combination.

Brief description of the drawing Figure I shows the DNA construct encoding the promoter, particle formation sequence and selection gene. (described in Examples 3/4).

Figure ■ shows the promoter, epitope with complete HB-S-Ag and selection site. A DNA gene construct encoding the gene is shown (described in Example 3/18).

Figure ■ shows promoters, epitopes with particle former residues, and selected genes. The encoding DNA construct is shown (described in Example 3/21).

Figure ■ shows the AST value of chimpanzee l during Hepa-Care treatment (Example 10/l).

Figure ■ shows the antigen levels of chimpanzee l during Iepa-Care treatment (Example 1O/! ).

Figure ■ shows liver enzyme A of chimpanzees boosted three times with Hepa-Care. TL and GGT values are shown (described in Example 1O/2).

Figure ■ shows the liver enzymes ALT and AST of chimpanzee 2 during Hepa-Care treatment. and GGT values and antigen values (described in Example 1O/3).

Figure ■ shows liver enzymes measured in untreated control chimpanzees (Example 1 (described in 0/3).

Figures ■ and Indicates body price (described in Example +1).

Figures XI and X■ show the antigenic titer and and antibody titers (described in Example 11).

Figures X■ and and antibody titer (described in Example 11).

The invention will be explained in more detail by the following examples.

Example 1 1. Fractional precipitation of HBV proteins using polyethylene glycol (PEG) The upper groove of the producing culture was collected and divided into 2.400 portions. 144 in each part g of PEG 6000 (Serva) was added and stirred for 20 minutes at room temperature. The mixture was dissolved and further stirred at 4°C for 6 hours. GS 30-ta in a 500 mi container Centrifuge for 30 minutes at 9,000 rpm (15,000Xg) at 10°C in a The precipitate was separated by this. Collect the upper groove and add 144 g of PEG6000. and dissolved in the same manner as above. The solution was stirred at 4°C for 3 hours. Centrifuge 6 The precipitate was collected from this solution in the same manner as above, except that the reaction time was continued for 0 minutes.

Z gel chromatography The material obtained after PEG precipitation was redissolved in 20-PBS and A-5m (Bio gel chromatography on Rad). Column dimensions are 25X 100 The bed volume was 0 m and 480 WLl. In a typical fractionation procedure, PE Load 1.000 μg of G-precipitated HBV protein at lθ~15rd, It was then eluted using PBS at a rate of 6 drops/min (18rd/hour).

Fractions above 3 m were collected. HBV protein eluted in the first peak. collect The collected fractions were applied to a CsC1 gradient.

3. Sedimentation in CsC1 gradient Including the first peak in column chromatography on A-5m and pre-preparation Approximately 30 fractions containing the prepared HBV protein were collected in approximately 100 vessels. This solution Adjust the density to 1.30 g/cc with CsC1, then 5W27/280-tar. (Beckman) into a polyaromer test tube.

1.35 g/cc of CsC1 solution 4rR1 was lowered and 1.25 g/cc was added on top of it. Add solution 4d with a density of g/cc, then add solution 4- with a density of 1.20 g/cc. The gradient was set by layering. This gradient was adjusted to 28.00 Orpm at 10°C. The test was continued for 50 hours. After that, the gradient was fractionated and the density was 1.20 g/cc. The purified HBV protein floating in the layer was collected. against water in the dialysis bag. The solution was desalted by three rounds of dialysis.

Example 2 Quantification of HBV protein 1. Quantitative AUSR [A If-125r sandwich by radioimmunoassay] In the Bch J radioimmunoassay (commercially available from Abbott), B Beads coated with guinea pig antibodies (anti-HBs) against hepatitis surface antigens were incubated with serum, plasma or purified protein and appropriate controls. Ta.

Any HBsAg that may be present was bound to the solid phase antibody. Aspirate unbound material and After washing the beads, human 1261-anti-HBs was allowed to react with the antibody-antigen complex on the beads. I responded. The beads were then washed to remove unbound +2J-anti-HBs.

)-Anti-HBs)lBsAg )-Anti-HBs-HBsAg　”'I-Anti-HBs)-Anti-HBs　・HBsAg- 'I - Gamma scintillation counter to remove remaining radioactivity on anti-HBs beads. Counted in the tar.

2. Quantification by ELISA Enzygnost HBsAg Tracer Sano Deutsch Assay (Behrin Wells were coated with anti-HBs (commercially available from G.G.).

Add serum, plasma or purified protein and appropriate controls to wells and incubate. I did it. After washing, the peroxidase-labeled HBSAg antibody is allowed to react with the remaining antigenic determinants. I responded. Unbound enzyme-conjugated antibody is removed by washing, and the enzyme on the solid phase is removed. Activity was measured. The enzyme-catalyzed reaction between hydrogen peroxide and chromogen is stopped by adding dilute sulfuric acid. I made it stop. The intensity of the color is proportional to the HBsAg concentration in the sample, and the HBsAg concentration is unknown. Spectroscopic comparison of the color intensity of the sample with that of the accompanying positive and negative control sera was obtained.

Example 3 Genetic constructs of the invention comprising a promoter, the desired antigenic sequence and a selection gene Preparation.

1. Isolation of MT promoter Plasmid pBPV-342-12 (ATCC37224) was endonucleated. Digested with ZeBglI and BamHI. Three DNA molecules were generated. Breaking focus The fragment is 4.5 kb and contains the metallothionein promoter and pBR322 sequence. and is easily distinguishable from the other fragments (2, Okb and 7.6 kb).

0.5 μg/μf containing 100 units of each restriction enzyme in a total volume of 200 μl of reaction buffer. Reactions were performed at the final concentration of DNA. Digestion is completed at 37°C for 3 hours. Agarose gel electrophoresis on 0.8% agarose gel after incubation of Confirmed by motion. The reaction was stopped by adding 4 μl of 0.5 MEDTA.

The 4.5 kb fragment was separated from other fragments by preparative 1.2% agarose gel electrophoresis. I let go. Elute the DNA from the agarose gel onto DE-81 Whatman filter paper. The DNA was removed from the filter paper in a high salt buffer.

DNA was purified by phenol-chloroform extraction and two ethanol precipitations. .

2. From the linked HBV-containing DNA of the 1.8 kb fragment encoding the HBV core antigen A 1.8 kb BamH [-BamH] fragment containing the BV-7 accord region was isolated. Ta. This fragment was converted into a 4.5 kb fragment containing the MT promoter and pBR residues (described in l). ).

Mix 2 μβ of the 1.8 kb fragment with 3 μl of the 4.5 kb fragment and add 2 units of DNA Incubate at 14°C in a total volume of 10 μl of ligation buffer containing gauze and 2mM ATP. I connected it in the evening.

This ligation mixture was added to 150μ of a competent bacterial cell suspension for DNA uptake. added to. After DNA uptake, add LB agar plate containing 50μl/- of ampicillin. Spread bacterial cells onto the plates at a volume of 50-300 μβ of cell suspension per plate. I erased it. The agar plates were incubated overnight at 37°C. one isolated Screen bacterial colonies for the presence of plasmids containing the desired fragment. did.

3. Screening for bacterial colonies containing the desired plasmid. It was taken out with a toothpick and transferred to a test tube (5) containing LB-ampicillin medium. this test The tubes were incubated overnight at 37°C under a rapid shaking environment. Each grown bacterial suspension A liquid mini-plasmid preparation was made. Restriction endonuclease of various generated DNA This was confirmed by digestion with Rease Bgl. Two molecules, a 400 bp fragment and a 5 ．． I expected it to be a 9kb fragment. Digests were analyzed by agarose gel electrophoresis.

Plasmid DNA was isolated from bacterial cells.

4. Insertion of neomycin selection marker Restriction of the plasmid obtained from step 3 above It was linearized by digestion with the enzyme EcoR. Total volume of 50μl and lμg/ Reactions were performed at a final concentration of μl plasmid DNA. 50 units of EcoR [] and incubation at 37°C for 3 hours followed by agarose gel electrophoresis. Digestion was confirmed by electrophoresis. 1μ! Stop the reaction by adding 0.5M EDTA. Stop and use 3-4 volumes of ethanol with a final concentration of 0.3M sodium acetate. The DNA was precipitated at -80°C for 30 minutes. Add 50 μl of the precipitated DNA to Dissolved in distilled water.

2 μl of linear plasmid was added with metallothionein promoter and neomycin selection. DNA fragment containing the gene [Plasmid pMT-neo-E (ATCC/Exog 3.9 by digestion with the endonuclease EcoRI from kb fragment] was mixed with 3μβ and ligated together. single thin Bacterial colonies were screened for the presence of the desired plasmid.

5. Isolation of fragment containing U2 promoter sequence Plasmid pUC-8-42 (Ex (available from Ogene) was digested with the restriction endonucleases EcoRI and Apa. It became. Two DNA molecules were produced.

The fragment of interest contains the U2 promoter with 340 bp, and the other fragment (3 160 bp).

0.5 μg/μl containing 100 units of each restriction enzyme in a total volume of 200 μl reaction buffer. Reactions were performed at a final DNA concentration of . Completion of digestion was achieved by a 3-hour incubation at 37°C. After incubation, perform agarose gel electrophoresis in a 0.7% agarose gel. I confirmed it more. The reaction was stopped by adding 4 μl of 0.5M EDTA. tone A 340 bp fragment was extracted from the plasmid DNA by 1.2% agarose gel electrophoresis. Separated from A. Transfer DNA from agarose gel onto DH-81 Whatman filter paper. The DNA was eluted from the filter paper in a high salt buffer. Phenol/ DNA was purified by chloroform extraction and two ethanol precipitations.

6. Insertion of promoter sequence-containing fragment into polylinker plasmid Polylinker sequence (contains the following restriction sites: EcoR1, 5ack, Sma[.

Ava[, BamH[, BglI[, 5alt, Pstl, HindI[) Plasmid psP165 containing (available from Promega Biotec ) was linearized using the restriction enzyme EcoR [. lμg in a total volume of 50μl Reactions were performed at a final concentration of /μl plasmid DNA. 50 units of EcoR[ and incubate for 3 hours at 37°C. Digestion was confirmed by pneumophoresis. Start the reaction by adding 1 μl of 0.5M EDTA. Stop using sodium acetate to a final concentration of 0.3 M and 3-4 volumes of ethanol. DNA was precipitated at -80°C for 30 minutes. Add the precipitated DNA to 50 μl of distilled water. It was dissolved in

Combine 2 μl of plasmid DNA with 10 μl of DNA fragment containing the U2 promoter sequence. Mix and make a total volume of 25 liters containing 2 units of T4-DNA ligase and 2 mM ATP. Ligations were allowed to occur overnight at 14°C in μl of ligation buffer. Then, phenol/chlorine DNA was purified by loloform extraction followed by two ethanol precipitations and 1 0μ! of distilled water. The generated EcoR[ and Apal sticky ends were blunt-ended. I had to convert and connect the ends. mung bean nuku as follows Sticky ends were converted to blunt ends by reaction with Rease: DNA in reaction buffer (lμg/μl concentration) Add 20 units of enzyme to 25μl and add 1% glycerol. Give a final concentration and a final reaction volume of 35 μl. Incubate for 30 minutes at 30℃ tion, by phenol-chloroform extraction followed by two ethanol precipitations. The DNA was purified. This DNA was dissolved again in 5 μl of distilled water. The resulting smooth finish end containing 10 times higher T4 licase and 2 d ATP than that used above. Ligate together overnight at 14°C in a 15 μl reaction.

This ligation mixture was added to a 150 μl competent bacterial cell suspension for DNA uptake. added to the liquid. After DNA uptake, LB agar containing 50 μl/- ampicillin Place bacterial cells onto the plate in a volume of 50-300 μl of cell suspension per plate. Smeared. The agar plates were incubated overnight at 37°C. single isolation bacterial colonies were tested for the presence of a plasmid containing the desired U2 promoter fragment. and screened. The resulting plasmid was isolated from bacterial cells and digested with restriction enzymes. Characterized by analysis.

7. Synthetic oligo DNA nucleotide 89 (SEQ ID NO: 30) and MT promoter – Concatenation with fragment (4,5kb) A 4.5 kb fragment containing the MT promoter and pBR residues (described in l) was synthesized with a synthetic oligo It was ligated together with nucleotide 89 (SEQ ID NO: 30). This ligation mixture Added to 150 μl of competent bacterial cell suspension for DNA uptake. single Screen one isolated bacterial colony for the presence of the desired plasmid did. The new plasmid was digested with the endonucleases EcoR and Xba. I confirmed it more. Two molecules were expected, one of 2, Okb and one of 2.6kb. .

8. Synthetic oligonucleotide 101 (SEQ ID NO: 32) and plasmid (described in 7) connection) The plasmid (described in 7) was digested with BglI[ and BamH[, and the 13 nucleotide The fragment was taken out (described in l). First oligonucleotide 89 (SEQ ID NO: The generated fragment containing It was linked to Tido101 (SEQ ID NO: 32). After DNA uptake, single cells Screened for desired plasmids. Endonucleate the new plasmid Confirmed by digestion with EcoRI and Xbal or EcoRI and BgllI. I chewed it.

9. Synthetic NA oligonucleotide 99 (SEQ ID NO: 31) and 4.5 kb fragment ( (described in 1) The 4.5kb fragment (BglIf-Bam)11) was converted into a DNA oligonucleotide. 99 (SEQ ID NO: 31). Single bacterial colony containing various NAs After screening, the desired plasmid was digested with EcoRI (1,9 kb and 2 ．． (generating two 7kb fragments) and positive linearization with BglII or BamH. was characterized.

This new plasmid was then digested with Pstl and BamH. pBR residue, M 2.6 kb fragment containing the T promoter and oligonucleotide, and 2. Okb Two molecules of pBR residues were expected. A 2.6 kb fragment was isolated.

From the 2.6 kb fragment of the plasmid described in 10.9 and the plasmid (described in 8), Connecting separated fragments Containing DNA oligonucleotides 89 and 101 (SEQ ID NO: 30 and 32, respectively) The plasmid (described in 8) was digested with Pst[ and BglII. 2 pieces are expected It was. One is a 2.5 kb fragment containing pBR residues and MT promoter; was a 2.2 kb fragment containing pBR residues and both oligos.

This 2.2 kb fragment was combined with the pBR residues, MT promoter and oligonucleotide described in 8. 99 (SEQ ID NO: 31).

After screening for the desired plasmid, it was transformed into BgIn-Xba [ Characterized by restriction endonuclease digestion using oligo dna nucleo A 270 bp fragment of Tide and a 4.5 kb fragment of MT promoter and pBR. Fragments were expected.

11, 2.3 kb HBV BglI [-BglI [ligation of fragment HBV-containing D From NA, 2.3 kb including HBV BR $1, BLE S2 and S Bgl II-Bgl I [fragment isolated. This 2.3kb fragment was linked to a 4.5 kb fragment containing the thionein promoter (obtained as described in 1). I tied it up.

Mix 2 μl of the 2.3 kb fragment with 3 μl of the 4.5 kb fragment and add 2 units of T 4-1) In a ligation buffer with a total volume of 10 μm containing NA ligase and 2 mM ATP. Ligation was allowed to occur overnight at 14°C.

This ligation mixture was added to a 150 μl competent bacterial cell suspension for DNA uptake. added to the liquid. After DNA uptake, 50μl/rtd! L containing ampicillin B. onto agar plates with a volume of 50-300 μl of cell suspension per plate. Cells were smeared. The agar plates were incubated overnight at 37°C. isolated Screen a single bacterial colony for the presence of a plasmid containing the desired fragment. I did a ning.

12.8 Conversion of a portion of the HBV gene sequence by the BV core epitope The plasmid obtained from Ta. Two molecules are expected, one a 550 bP fragment and one a 6.25 kb fragment. The 6.25 kb fragment was isolated after agarose gel electrophoresis.

A 6.25 kb fragment was inserted into the IO encoding the epitope portion of the HBV core gene. 270 bp fragment of the plasmid (same as above, with glI[ and Xba[ After digestion and isolation of the fragments).

This ligation mixture was added to a 150 μl competent bacterial cell suspension for DNA uptake. added to the liquid. Isolate a single bacterial colony for the presence of the desired plasmid Screened. The new plasmid was verified by digestion with BamHI. 9 Three molecules were expected: 50 bp, 450 bp and 5.150 bp fragments.

13. Preparation of “vehicle” plasmid The plasmid (described in 11) was digested with EcoRI and Xba. 2.450 Two molecules, a bp fragment and a 4.350 bp fragment, were expected, and after gel electrophoresis, 4.3 A 50 bp fragment was isolated.

, :) 4.350 bp fragment was inserted into the HBv-8 gene up to the ATG Kara xba1 site. A DNA oligonucleotide that encodes the entire DNA sequence of the child, with ATG changed to ATA. It was linked to cleotide 39 (SEQ ID NO: 29).

14. The core epitope upstream of the complete HBV-8 gene and then this “vehicle” When the plasmid was digested with Pstl and Xbal, 600 bp plasmid residue and Two molecules of the 3.850 bp fragment were expected and the latter fragment was isolated and 2,800 bp (2,700 bp) Ps isolated after plasmid digestion It was ligated with the tl-Xbal fragment.

After screening for the desired plasmid, it was incubated with EcoRI and Xba I, Restriction endonuclease quenching using EcoRr and BglI [and BamHI] Characterized by .

+5. Insert selection marker The plasmid (described in 14) was linearized with EcoRr. 50μ! total of Reactions were carried out at a final concentration of 1 μg/μl plasmid DNA. . Add 50 units of EcoR and incubate for 3 hours at 37°C. Digestion was confirmed by low gel electrophoresis.

The reaction was stopped by the addition of 1 μl of 0.5M EDTA and the final concentration was 0.5M. 30 minutes at -80°C using 3M sodium acetate and 3-4 volumes of ethanol. DNA was precipitated. The precipitated DNA was dissolved in 50 μl of distilled water.

2 μl of linear plasmid was added with metallothionein promoter and neomycin selection. DNA fragment containing the gene (described in 4) 3μ! and were combined together. single Screen one bacterial colony for the presence of the desired plasmid, plus Mido was isolated and characterized.

After digestion with EcoRl and BglII, each of the above-mentioned gene constructs was digested with MT promoter. The protein-containing DNA fragment was isolated after digestion with EcoRI and BglII. By replacing the U2 promoter-containing DDNA with It can also be produced using a motor.

16. Escherichia coli xanthine guanine phosphoribosyltransferase (egpt ) Isolation of selected genes After digesting plasmid pMSG with Bam) I and Bgl I, egpt selection was performed. A fragment (1,8 kb) containing the alternative gene was isolated, and the 4 fragment containing the MT promoter was isolated. ．． It was ligated with a 5kb fragment (BglI [-BamHI, described in 1).

After screening for the desired plasmid, the plasmid is isolated and purified. , and was completed by converting the old Bam site to the EcoR1 site.

17. Isolation of desired DNA sequence by PCR method One DNA fragment (400bp) was isolated after gel electrophoresis. The fragment is used as a primer for a specific oligonuclease. PCR method (Example 5) using leotide 131 and 132 (SEQ ID NOs: 33 and 34) (described in ).

This DNA fragment was digested with endonucleases Bamold and XbaE, and then It was purified by electrophoresis. The isolated PCR fragment was transformed into a plasmid (described in 13) was ligated with a 6,25 kb fragment isolated after digestion with BglI and Xba. Closed. After DNA uptake and bacterial transformation, a single bacterial colony was Screened for lasmids.

18. Insert selection marker By adding a selection gene (described in 15) to a plasmid (described in 17), Completed Lasmid.

Isolation of the promoter in 19, 82 EcoRI-Bgl II from pSP65H2 (available from Exogene) The promoter was isolated in 82 as a fragment (2 kb).

In all constructs described above, all promoters are linked to EcoR[/BglII fragment It can be exchanged as

20. Conversion of part of the 8BV gene genetic sequence The plasmid obtained from II above was treated with endonuclease BgllI and Xba Digested with I. Two molecules are expected, one of which is a 6.250 kb fragment; The fragment was isolated after agarose gel electrophoresis.

6. Link the 250 kb fragment with oligo DNA nucleotide 23 (SEQ ID NO: 28). I tied it up. Transfer this ligation mixture to 150 ul of competent DNA uptake. added to the bacterial cell suspension. Combine a single isolated bacterial colony with the desired plasmid. Screened for presence. Transfect the new plasmid with endonuclease εco This was confirmed by digestion with RI and BglII. One is 1.9kb and one is 4.45 Two molecules of 0 kb were expected.

21. Insertion of egpt selection marker The plasmid (described in 20) was linearized with EcoR. 100μl Perform reactions at a total volume and final concentration of 0.6 μg/μl plasmid DNA. did. After adding 60 units of EcoRI and incubating for 3 hours at 37°C. Digestion was confirmed by agarose gel electrophoresis.

The reaction was stopped by the addition of 2μβ of 0.5M EDTA and a final concentration of 0.5M EDTA was added. Incubate the DNA with 3M sodium acetate and 4 volumes of ethanol for 1 hour at -80°C. precipitated. The precipitated DNA was dissolved in 50 μl of distilled water.

Add 2 μl of linear plasmid to metallothionein promoter and egpt selection gene. DNA fragment (3,7 kb) containing the child (described in 16) Mix with 3 ul and connect together. I tied it up. Screen single colonies for the presence of the desired plasmid did. Each gene construct listed in Table 1 can be prepared using the same method as above.

Example 4 Transfection of mammalian cells using the constructs of the invention In order to obtain secretion of significant amounts of HBV peptides encoded by The DNA construct must be used to transfect mammalian cells. . in two steps (i.e. separate transfections for each construct) or in one step. simultaneous transfection (i.e., one transfection using preparations of image constructs) infection was performed. For the use of different selection markers on two constructs or by detecting the secretion of the expression product of the image construct by immunoassay. The transfection was confirmed at the time.

Alternatively, a sequence encoding an HBV peptide sequence of the invention and a complete S or or another sequence encoding an HAV protein in a single construct. can be set.

Example 5 Polymerase chain reaction (PCR) The polymerase chain reaction is used to isolate specific DNA nucleic acids in selected regions of known genomic sequences. It is possible to amplify the otide sequence more than a million times in vitro (Thomas J., White, Norman Arnleim, Henry A.

Erich 1989: The polymerase chain re action, Technical Focus.

Volume 5, No. 6; S, Kwok and R, Hjguchi 1989: Avo idingfalse positives with PCR, Nature , Vol. 339, pp. 237-238).

DNA isolated from cells or plasmid DNA is treated to separate its complementary strands. Let go. The strands are then combined with X nucleotides (where X is typically 50 to 2.00 chemically synthesized to match sequences separated by 0 base pairs) Anneal with an excess of two DNA oligonucleotides (each 20-25 base pairs long). Let me know.

These two oligonucleotides have a sequence matching the two oligonucleotides. In vitro DNA synthesis catalyzed by DNA polymerases that replicate DNA between Serves as a specific primer for

If the two primer oligonucleotides contain the correct sequences, the 5' and 3' It is possible to create new digestive sites.

After multiple reaction cycles, a large number of DNA fragments of the desired length are obtained, which can be run on a gel. Purified by electrophoresis and characterized by restriction enzyme digestion and agarose gel electrophoresis. Signed. The amplified purified DNA fragment is ligated to another fragment, that is, a plasmid. I used it to

PCR-DNA fragments were amplified with blunt ends. To give a sticky end (ligation operation) (for production), the fragment must be digested with the desired endonuclease and purified again. No.

The PCR reaction will run for 20-30 cycles. l cycle is a different reaction Divided into 3 stages by time and different reaction temperature (controlled by PCR thermocycler) It will be done. The first step is "denaturation" of the template DNA (1 minute/95°C), and the second step is [Hybridization J (1 minute 155°C)] of template DNA and primer The third step is "polymerization" (2 minutes/72°C).

The final volume for one assay is e.g. 30 μl, which includes the final concentration of M: PCR buffer (IX), nucleotides containing 200 μM each of the four types of nucleotides Otide mixture, 200 ng of each of the two primers per 301ti, aq ua bidest 0.5 units of Taq polymerase per 30 μl.

Example 6 Culture recipient cells of transfected cells secreting protein (obtained from ATCC) Cl27 or CHO cells) in standard growth medium (D MEM + 10% fetal calf serum, glucose and glutamine) (1 -2　XIO@cell/dish, φ10an).

The next day, remove the medium and 4 hours before adding the DNA precipitate onto the cells). Washed twice with BS. Then, DMEM S- not containing FC3 was added and left for 4 hours. A DNA precipitate (prepared as described below) was then added to the cells. 4 hours later again Remove the medium and add a 3-glycerol mixture (50 yJ of 2XTBS buffer, 30 rnl of cellol and 120 rnl of distilled water were added. At 37°C for 3 minutes. After incubation, the glycerol mixture is immediately removed and the cells are Washed with XPBS. Cells were cultured overnight in DMEM 8- containing 10% FC8. Ta.

After 48 hours, trypsin-EDTA solution (0,025% trypsin + 1mMED Cells were harvested from the dishes by treatment with TA). Then trypsin-ED Cells were washed with IXPBS to remove TA and DMEM containing 10% FCS. and dispensed into 24 Costa-well plates (cells from one dish cells into four 24-well plates).

When the cells had grown sufficiently, a selective medium was added [e.g., at a concentration of 0.5 to 1/- neomycin or xanthine (250 μs/rd), hypoxanthin (1 5 μg/ml) or adenine (25 μg/1ni), thymidine (10 μg /m/), aminopterin (2 μg/ynl), mycophenolic acid (25 μg /rd)). The first proliferating cell colonies were observed after 2 weeks.

10 μg of plasmid DNA and 20 μg of carrier DNA (salmon sperm DNA, 1 calf) Thymus DNA), Tε buffer (10mM Tris-HCI, 1mM ED) T.A.

pH 7.05) to a final volume of 440 μl and 60 μl of Mixed with 2M CaC1z. Then equal amount of 2 x TBS (Hepes 50 mM, NaCl280mM, NaJP041.5mM, pH7.05) Add and mix well. Incubate the precipitation solution at 37°C for 30 minutes and and directly added to the cells to be transfected.

Example 7 Preparation of adjuvant for purified particles Add 1:10,000 volumes of antigen at the desired concentration suspended in sterile saline solution. Melopur, 1/10 volume filtration sterilization method 0.2M KAI (SO-)!・12H 2O was added. Adjust the pH to 5.0 with sterile IN NaOH and suspend the suspension. The mixture was stirred at room temperature for 3 hours. Alum precipitated antigen at 2.00 rpm for 10 minutes Collected by centrifugation between Resuspend in saline and divide into aliquots under sterile conditions.

Cell supernatants of HB cocoagen-secreting cells were collected and concentrated by ultrafiltration. This concentration The material was heated in a Beckman 5W280-tar at 4°C at 20.00 rpm for 15 minutes. Clarified by centrifugation for 1 minute.

Beckman 5W280 at 28.00 rpm for 24 hours at 4°C in one temp. Particle formation was tested by sucrose density gradient centrifugation (0-45% sucrose). the slope It was fractionated and each fraction was analyzed by EL [SA].

Example 9 The table below gives some results of the EL[SA analysis described below for the immunogenic particles of the invention. .

Table ■ shows anti-Bre S1 monoclonal antibody MA18/7 and anti-HBs monoclonal antibody HBV sequences of the present invention containing the pre-S1 epitope and S region using antibody G022. ELISA table of purified HBs antigen particles produced from column constructs shows CsC1 density Fractions (21) collected after the gradient are shown.

Table IV-2 Table V shows polyclonal antibodies against HB Cocoa and monoclonal antibodies against HB-S-Ag. produced from any HB cocoa train construct of the invention using local antibody G022. EL [SA data of purified purified HB cocoa raw particles are shown.

Example 10 Administration experiment of Hepa-Care in chimpanzees Hepa-Care was Presents hepatitis B surface antigens (Sl and S) in a fixed ratio (50:50) It is a particle that is used to treat chronic carriers of hepatitis virus.

experiment l Hepatitis B preserved chimpanzee 1 was transferred to No. 0. 18μ per injection at 4 and 8 weeks treated with Hepa-Care (intramuscularly) at a dose of 100 g.

Liver enzymes (Panel ■) as well as hepatitis B antigen levels (Panel V) were monitored.

Experiment 2 Chimpanzee 1 was boosted at weeks 30.34 and 38 after the above treatment.

The results are shown in Figure ■.

Experiment 3 Chimpanzee 2 was treated with Hepa-Care. However, what is chimpanzee 1? Differently, given intravenously. The dose was 2■. The results are shown in Figure ■.

Liver enzymes were also monitored from control chimpanzee 3, and the results are shown in Figure ■.

Example 11 Hepa-Care treatment: (See Example IO for definitions) Patient 1 (male, age 65 years, medical history) 2 years): Hepatitis B parameters: HBsAg positive, anti-HBs negative HBeAg negative Anti-HBe positive Anti-HBc negative 0. 1. Treatment with Hepa-Care at 6 and 7 months (i, m,) L, ta.

The results of antigen and antibody measurements are given in Figures ■ and X.

Patient 2 (female, age 48 years, medical history 12 years): B-hepatitis parameters: HBsA g Positive HBeAg Negative Anti-HBs negative Anti-HBe positive Anti-HBc positive 0. Treatment with Hepa-Care at I and 6 months (i, m,) L, . The results of antigen and antibody measurements are given in Figures XI and X■.

Patient 3 (female, age 41 years, medical history 5 years): Hepatitis B parameters: HBsAg Positive HBeAg Negative Anti-HBs negative Anti-HBe positive 0. Treatment with Hepa-Care at I, 2 and 5 months (i, m,) Ta.

Measured values for HBs antigen and anti-HBs antibody are shown in Figures X and XIV.

Sequence number: 1 Sequence type: Nucleotide Sequence length: 558 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: PCR amplification λτGG input CATT GACCCT TAT AAA GAA TTT GGA GCTACT GTG GAG TTA CTCTCG TTT TTG C CT TCT GACTTCTT CCT TCCGTA CCA GAT CTCCTA GACACCGCCT CA GCT CTG 7. %T CGA G decrease GCCTTA GAG TCTCCT GAG CAT TGCTCA CCT CACCAT ACT CCA CTCAGG Trowel GCCATT CTCTGCTCG GGG G8 TTG ATGλCT CTA GCT ACCTG G GTG GGT decrease T λx'r TTG C)-1GAT C CA GCA TCCACA GAT CTA GTA GTCλλτ TλT GTT decrease T ACT AACATG GGT TTA , IAG ATCAG G C Iris CTλ Ding TG TGG TTT CAT ATA TCT TGCC TT ACT TTTGGλ AGA GAG ACT GTA CTT GA A TAT TTG GTC τCTττCGGA GTG TGG ATT C GCACT CCT CCA GCCTATAGA CCA CCA Enter AT GCCCCT ATG TTA TCA ACA CTTCCG G touch ACT ACT GTT GTT AGA CGA CGG GACC AGGCAG G TCCCCT AGA AGA AGA ACT CCCTCG CCTC GCAGA CGT AGA TCT CAA TCG CCG CGT CG CAGAAA TCT CLI TCT CGG CAA TCT CAA TGT TAG SEQ ID NO: 2 Sequence type: Nucleotide Sequence Length: 504 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: PCR amplification ATG GACλττ CλCCCT τλτ AAA Oλ enter DingτT GC A GCTACT GTG GAG TTA CTCTCG TTT TTG CCT TCT GACTTCTTT CCT TCCGTA CGA GAT CTCCTA QACACCGCCTCA GCT C'': G TAT CG A GAA GCCTTA GAG TCTCCT GAG CAT TCCT CA CCT CACC AT ACT GCA CTCAGG C)A GCC ATT CTCTCCTCG GGG GAA TTG ATGλCT CTA GCT ACCTGG GTG GGT AAT AAT ττGCAAGA T CCA GCA TCCAGA (aλTCτλ GTA GTCAAT TATGττ AAT ACT AACATG GGr TTA AAG AT CAGG CAACTA TTG TGG τττ CλTAτλ TCT T GCCTT ACT τττGGλ A CA GAG ACT GTA CTT CAA TAT TTG GTCTCTTTCGGA GTG TGG AT T CGCACT CCT CCA GCCTATAGA CCA CCA ) 2T GCCCCT ATG TTA TCA ACA CTTCCG G ACT ACT GTT GTT AGA CGA CGG GACCGAGG CAGG TCCCCT ACA λHAλGA ACT CCCTCG CCT CGCACA CGT Sequence number: 3 Sequence type: Nucleotides Sequence length: 504 b[ ) Number of chains Knee end chain Topology Straight chain Sequence type/Genomic DNA Origin: HBV core Direct origin: PCR amplification ATG (a in CλτTQλCCCT τλτ λ in GAA TTT G(, λ GCTACT GTG GAG dingτA CTCTCG TTT TTG this CT TCT GACTTCTT CC T TCCGIA 00 people Gλ TCτCCTA GACACCGCCTCA GCT CTG TAT CCA C input λ GCCττAGλG TCTCCT GAG CAT TCCTCA CCT CACCAT ACT GCA CTCAGG CAA GCCAT T CTCTGCTGG GGG CAA TTG ATGACT CTA G CT ACCTGG GTG GGT AAT AAT TTG O person GAT CCA GCA TCCACA CAT CTA GIA GTCλ admission T ATGTT λ entry ACT entry CATG GGT TTA AAC entry τCA GG CAACTA TTG TGG TTT CAT ATA TCT TG CCTT ACT TTTGGA AGA GAG ACT GTA CTT GAA TAT TTG GTCTC TTτCGGA GTG TGG Inputττ CGCACT CCT CCA GCCτλTAGA CCA CCA λ Insertion GCCCCT ATCττλ TCA λCACττCCG GAA ACT Enter CT GTT GTT ACA CGA CGG CACCGAGGCA GG TCCCCT AGA AGA AGA ACT CCCTCG CCT CGCA(, enter CCT Sequence number: 4 Sequence type: Nucleotides Sequence length: 534 lap Number of strands End chain topology: straight chain Type of Sequence DNA Origin: HBV core Direct origin: PCR amplification TCCAACCTG TGCCTT GGG TGG CTT TGG GGC ATG CACATT GACCCT TAT AAA QAA TTT GG A GCTACT GTG GAG T TA CTCTCG TTT TTG CCT TCT (aCTTCTTT CCT TCCGIA α included GAT C TCCTA GACACCGCCTCA GCT CTG τλτ C(aA QAA GCCTTA GAG TCTCCT GAG CAT TGCTCA CCT CACCA 'l' GCA CTCAGG CM GCCAT T CTCTGCTG GGG Shigeru TTG ATGACT CTA GCT ACCTGG GTG GGT AAT AAT ττGCAAGAT CC A GCA TCCACA GAT CTA GIA GTCAAT TATG TT AAT ACT AACATG GGT TTA AAG ATCAGG CAACTA TTG TGG TTT CAT ATA TCT TGCC TT ACT TTTGGA AGA GAG ACT GTACTT CAA TAT ττGGτCTCTττCGG A GTG TGG ATT CGC ACT CCT CCA GCCTATAGλ CCA CCA AAT GC CCCT ATG TTA TCA ACA CTTCCG GAA ACT ACT GTT GTT AGA CGA CGG GACCGAGGCAGG TCCCCT AGA AGA AGA ACT CCCTCG CCTCG CAGA C0 sequence Sequence number: 5 Sequence type: Nucleotide Sequence length: 534 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: PCR amplification TCCAAACCTG TGCCTT GGG TGG CTT TGG GGC ATG GACATT GACCCT TAT AAA GAA TTT GQ A GCTACT GTG GAG TTA CTCTCG TTT TTG CCT TC T GACTTCTT CCT TCCGTA CGA GAT CTCCTAGACACCGCCTCA GCT CTG TAT CGA GIA GCCTTA CAG TCTCCT GAG CAT TGCTCA CCT CACCAT ACT GCA C TC71- GG CAAGCCA TT CTCTGCTGG GGG 鞞 TTG ATGACT CTA GC T ACCTGG GTG GGT AAT &1l-T TTG CAAGA T CCA GCA TCCA(aA aT CTA GTA GTCAA T TATGTT AAT ACT AACATG GGT DingτA kkG AT CAGG CAACTA TTG TGG TTT CAT ATA TCT TGCC″rT ACT TTTGGA AGA GAG ACT GTA C TT (aA TAT TTG GTCTCT: τCGGA GTG TGG Entering τT CGCACT CCT CCA GCCτλTAGA CCA CCA Entering λT GCCCCT ATCττλ TC A ACA CTTCCG CA A ACT ACT GTT GTT AGA CGA CGG CACCGA GGCAGC; TCCCCT ACA AGA AGA ACT CCCTC G CCTCGCAGA CGT Sequence number: 6 Sequence type: Nucleotide Sequence length: 87 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin Bichemical synthesis ATCCTCTGCTGG GGG GAA TGG ATG ACT CTA GCTACCTGG GTG GGCAAT AAT TTG GAA GA T CCA GCAτC Ding AGCGACCTT GTA GTA AAT Sequence Number Near Sequence type: Nucleotides Sequence length: 81 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin :HBV core Direct origin: Chemical synthesis GACATT GACCCT TAT) Sequence length: 114 bl) of chain Several knee-end strands Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: Chemical synthesis TCCjLACCTCTGCCTT GGG 'rGG CTT TGG GG CλτGCAC ττ CACCCT TAT λ 0 TTT G( , λ GCT A CTGTG GAG TTA CTCTCG TNT TTG CCT TCT (,ACTTCTTT CCT TCCGTCAGG Sequence number = 9 Sequence type: Nucleotide Sequence length: 90 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin :Chemical synthesis GAT CTCCTA CACACCGCCTU GCT CTCτλTCC AA GCCTTA GAG TCT CCT GAG CTA TGCTCA CCTC input COτ input CT GCA CTCAGG CAA GGT Sequence number: 10 Sequence type: Nucleotide Sequence length: 261 bp Number of strands Knee end strand Topology :Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: Chemical synthesis ATCGj', CATT GACCCT TλTAAA ω■ TNT G■, GCTACT GTG αG TTA CTCTCG TTT TTG CCT TCT GACTTCTT CC T TCCCTCAGG GAT CTC CTA GACACCGCCTCA CCT CTG TAT CGA 0 people GCCTTA GAG TCTCCT 榔GCτλ TGCTα CCT α CCA'r ACT Ghi CτCλGG CAA GGT ATCCTCTG CTGG GGG GAA TGG ATGACT CTA GCT ACCT GG GTG GGCAAT AAT TTG CAAGAT CCA GCA TCT,! 1. GG CACCTT GTA GTA AAT Sequence number = l l Sequence type: Nucleotides Sequence length: 291 bp Number of strands Knee-end strand Topology: Linear Sequence type Nigatum DNA Origin: HBV core Direct origin: Chemical synthesis TCCAAACCTG TGCCTT GGG TGG CTT TGG GGC ATG QACATT GACCCT TAT AAA GAA TTT GG A GCTACT GTG CAG ττ CTCTCG TTT TTG CCT TCT GACTTCTT CCT TCCGTCAGG GAT CTCCTA GACACCGCCTCA GCT C"l'G TAT CG A GAA GCCTTA CAG TCTCCT GAG CTA TGCT CA CCT CACCAT ACT GCA CTCλGG CAA GGT ATCCTCTGCTGG GGG GAA TGG ATGACT CTA GCT ACCTGG GTG GGCAAT AAT TTG CAAGA T CCA GCA TCT AGG GACCTT GTA (JA AA T SEQ ID NO: 12 Sequence type: Nucleotide Sequence length: 123 bl) Number of strands Knee-end strand Topology :Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: Chemical synthesis ACCTGG GTG GGT AAT λλτ ττGCλλ GAT CC A GCATCCAGA GAT CTA GTA GTCAAT TAT G TT AAT ACTAACATCG GT TTA AACATCAGG CA A CTA TTG τGGτττ CAT ATA TCT TGCCTT ACT TTT SEQ ID NO: 13 Sequence type: Nucleotides Sequence length + 1381) p Number of strands Knee end strand Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct origin: Chemical synthesis GCA TCCAGA GAT CTA GTA GTCAAT TAT GT T AAT input Cτ AACATG GGT TTA AAG ATCAGG C AA CTA TTGTGG TTT CAT ATA TCT TGCCTT ACT TTT GGA AGAGAG ACT CTA CTT GAA TAT T TG GTCTCT ττCCGAGTG TGG SEQ ID NO: 14 Sequence type: Nucleotides Sequence length: 294 bp Chain Number of knee heavy chains Topology: Linear Sequence type Nigetum DNA Origin: HBV S 2 ayw/adw Direct origin: HBV DNA ATG CAG TGG AAT TCCACA ACCTTCCACCAA ACT CTGOλ included GAT CCCACA GTG AGA GGCCTG τλτ TTCCCT CCTGGT GGCTCC AGT TCA GGA λCAGτλ AACCCT GTT CτGAGT ACT GCCTCT CCCTTA TCG TCA input τCTTCTCC input GGλτλ GAG AACATCACA TCA GGA TTC-CTA CCA CCCCτT CTCGTG TTA CAG GCG G GG TTT TTCTTG TT G ACA AGAATCCTCACA ATA CCG CAG ACT C TA CACTCG TGG τGGλCT TCT CTCAAT TTT CTA GGG GGA ACT ACCGTG TGTCTT GGCCAA AAT TCG CACTCCTCA ACCTCCAATGACTCA C CA ACCTCT TCT CCT CCA ACT TGT CCT GG T τλTCGCTGG ATG TGT CTG CGG CCT TTT Input τCλτ 0 TTCCTCTTCATCCTG CTG CTA TCCCT CATCTTCTTG TTG GT”CTT CTG GACτλτ CAA GGT ATG TTG CCCCTT TCT CCτCTλ ATT C C”-GGA TCCTCA ACA ACCAGCAG GCA CCAT GCCGG ACCTGCATG ACT ACT GCT CAA GGA ACCTCTATG Tλτ CCCTCCTG TGCTGT ACCAA A CCT TCCGACGGA AAT TGCAC CTCT input τT CC CATCCCA TCA TCCTGGGCT TTCGGA AAA TτC Cτλ TCG Gj-TCG GCCTCA GCCCGτ TTCTCCT GG CTCACT ττ input CTA GTG CCA TTT GTTCA G TGG TTCGTA GGG CTT TCCCCCACT GTT TG G CTTTCA GTT ATX TCG ATG ATG 'rGG τλ T TGG GGG CCA AGTCTG TACAGCATCTTG AG T CCC dingττ ττλ CCG CTCTTACCA Enter ττ TTCττ τ TCT CTT TCG GTA TAC Enter τ SEQ ID NO: 15 Sequence type: Nucleotide Sequence Length: 99 bp Number of strands Knee heavy chain Topology Bilinear Sequence type Nigatum DNA Origin: HBV core Direct origin: Chemical synthesis τλτ GTT λλτ ACT AAC τG GGT TTA AAG A TCλGGCAA CTA TTG TGG TTT CAT ATA TCT TCCC7 ACTTTT GGA AGA GAG ACT GTA C τTGAA τλT ττGGτC SEQ ID NO: = 16 Sequence type: Nucleotide Sequence length: 390 bp Number of strands Knee heavy chain Topology: Linear Sequence type Nigetum DNA Origin: HBV core Direct Origin of: PCR amplification TCCAACCTG TGCCTT GGG TGG CTT TGG GGC ATG GACATT GACCCT τλT AAA GM TTT GGA GCTACT GTG CAG TTA CTCTCG TTT TTG C CT TCTGACTTCTTT CCT TCCGTA CGA GAT C τOCτA CACACCGCCTCA GCT CTG τλT CGA G AA GCCTTA GAG TCTCCT GAG CAT TGCTCA CCT CACCAT ACT G■、CτCλGG CAA GCCλττ CTCTGCTGG GGG GAA TTG ATGACT CTA GCT ACCTGG GTG GGT AAT AAT TTG CAACAT C CA GCA TCCAGA GA T CTA GTA GTCAAT TAT GTT AAT ACT Input λCATG GCT TTA AAG ATCAG G CλACTA TTCTGG TTT CAT ATA TCT TGCC TT Input Cτ TTTGGA AGA GAG ACT CTA CTT τAT TTG Gτ0 Sequence number = 17 Sequence type: Nucleotide and corresponding protein sequence length: 60 bp Number of chains: knee heavy chain Topology: bilinear Sequence type: Nigetum DNA Origin: HBV Sl ay Direct origin: Chemical synthesis AAT CCT CTG GGA TrCTrr CCCGAT CACCAG TrG GATCCA GCCTrCAGA GCA AACACCGCAA sn Pro Leu Gly Phe Phe Pro Asp His G in Leu AspPro Ala Phe Arg Ala Asn Thr Ala SEQ ID NO: 18 Sequence type: Nucleotide Length of protein sequence corresponding to: 63 bp Number of chains Knee heavy chain Topology: Linear Sequence type Nigetum DNA Origin 1f: HBV Sl ay Direct origin: Chemical synthesis CCT GCC CACCAAT CGCCAG TCA GGA AGG CAG CCTACCCCCG CTG TCT CCA CCT TTG AG A AACPro Ala Ser Thr Asn Arg Gin Ser Gly Arg Gin Pr.

Thr　Pro　Ile　Ser　Pro　Pro　Leu　Arg　Asn Column number: 19 Sequence type: Nucleotide and corresponding protein sequence length: 63 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Sl ay Direct origin: chemical synthesis GAT CCA GCCTrCAGA GCA AACACCGCA AAT CCA GATTGG GACTrCAAT CCCAACAAG GACAC CAsp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Dingrp Asp Phe Asn Pro Asn Lys Asp Ding hr Sequence number: 20 Sequence type: Nucleotide and corresponding protein sequence length: 108 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Sl ay Direct origin: chemical synthesis CCG CACGGA GGCC “n′′ Low G GGG TGG AGCCC T CAG GCTCAG GGCA A CTA CAA ACT TrG CCA GCA AAT CCG CCTCCT GCCTCCACCAAT CGCCAG TCA GGA AGG CAG CCTPro His Gl y Gly Leu Leu Gly Trp Ser Pro Gin Al aGln Gly Ile Leu Glu Thr Leu Pro Ala Asn Pro Pr.

Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg　Gin　Pr.

Sequence number: 21 Sequence type: nucleotide Sequence length: 63 bp chain number knee heavy chain topology digonal Sequence type Nigetum DNA Origin: HBV Sl ad Direct origin: chemical synthesis CCT GCCTCCACCAAT CGG CAG TCA GGA AGG CAG CC lower ACT CCCATCTCT CCA CCT CTA AG A GAC-X sequence number = 22 Sequence type: Length of nucleotide and corresponding protein sequence: 108 tap chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Sl ad Direct origin: chemical synthesis CCA CACGGCGGT A’mouth °” Low G GGG TGG AGCCC T CAG GCTCAG GGCAT nG ACCACA GTG TC A ACA ATr CCT CCTCCT GCCTCCACCAAT CG G CAG TCA GGA AGG CAG CCTPro His Gly Gly IIs Leu Gly Trp Ser Pro Gin Ala Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pr.

Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg　Gln　Pr.

Sequence number = 23 Sequence type: nucleotide Sequence length: 60 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV S2 ay Direct origin: chemical synthesis CAG TGG AAT TCCAGA ACCTrCCACCAA ACT CTGCAA GAT CCCAGA GTG AGA GGCCTG TAT -X sequence number: 24 Sequence type: nucleotide Sequence length: 60bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV S2 ay Direct origin: chemical synthesis GAT CCCAGA GTG AGA GGCCTG TAT TTCCCT GCTGGT GGCTCCAGT TCA GGA ACA GTA AA C-X sequence number: 25 Sequence type: nucleotide Sequence length: sss bp chain number knee heavy chain topology bilinear Sequence type Nigetum DNA Origin: HBV core adw Direct origin: PCR amplification ATCGACATT GACCCT TAT λAAGλλ TTT CGA GCTACT GTG GAG TTA CTCTCG TTT TTG CC T　TCT　GACττOTTT　CCT　TCCCTA　CGA　GAT　C TCCTA GACACCGCCTCA GCT CTG TAT CGA G M GCCTTA GAG TCTCCT GAG CAT TGCTCA C CT CACCAT ACT GCA CTCAGG CAA GCCATT CTCTGCTGG GGG (aA TTG ATGACT CTA GCT Input CCTGG GTG GGT MT AAT TTG Cλ Input GAT CC A GCA TCCAGA GAT CTA GTA GTC product τ τλTGT T AAT ACT MCATG GGT TTA AAG ATCAGCCA ACTλ TTG TGG TTT CAT ATA TCT TGCCTT ACT TTTGGA AGA GAG ACT GTA CTT GAA T AT TTG GTCTCTTTCGGA GTG TGG ATT CGCA CT CCT CCA GCCTATAGA CCA CCA Shin T GCCC CT ATG TTA TCA ACA CTTCCG GAA ACT Enter C T GTT GTT AGA CGA CGG GACCCAGGCAGG T CCCCT AGA AGA AGA ACT CCCTCG CCTCGCA GA CGT AGA TCT CAA TCCCCG CGT CGCAG AGA TCT CAA TCT CGG CAA TCT CM TGT τ λG sequence number: 26 Sequence type: nucleotide Sequence length: 678 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV S adw/ayw Direct (7) Origin: HBV DNA Zλ　CoG　AACATCACA　TCA　GGA　ττCCτA　α’A　C CCCTT CTCGTG ττλ CAG GCG GGG τττ TTC Ding τG TTG ACA AQA ATCCTCλO entering τλ CCG CA G AGT CTA CACTCG TGG TGG JLCT TCT CT CAAT TTT CTA GGG GGA ACT ACCGTG TGT CTT GGCCAA AATTCG CAG TCCTCA ACCTCCA AT CACTCA CCA ACCTCT TGTCCT CCA ACT TCT CCT GGT τλτ CGCTGG ATCTGT CTG CG GCGT τττ ATCATCTTCCTCTCATCCTG CTG C TA　TGCCTCλTCTTCττGT″rG　GTT　CTT　CTG　G ACTAT CAA GGT ATG ττGCCCGTT TGT CCT CTA ATT CCA GGA TCCTCA ACA ACCAGCACG GGA CCA TGCCGG ACCTGCATG ACT ACT GC T CAA GGAλCCτ0τ ATG TAτ CCCTCCTG TG CTGT ACCAAA CCT DingCGGACGGA AAT TGCACC TGT ATT CCCATCCCA TCA TCCTGGGCT TTCG GA Aj-A TTCCTA TGG GAG TCG GCCTCA GC CCGττTCTCCTGG CTC input Gτ TTA CTA GTG CCA τDing TGττ GAG TGGTTCCTA GGG CTT TCCCCC ACT GTT TGG CTT TCA GTT Aτλ GG ATG A TG TGG TAT TGG GGG CCA AGT CTG τ CAG CATCTTG AGT CCCTTT TTA CCG CTG TTA C CA ATT TTCTTT TGTCTT TGG GTA TλCλDing T arrangement Column number: 27 Sequence type: nucleotide Sequence length: 585 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV S adw/ayw Direct origin: HBV DNA cτ included GACTCG TGG TGG ACT TCT CTCAAT TT T CTA GGGGGA TCT CCCGTG TG’l” CTT GG CCAA AAT TCG CAG TCCCCA ACCTCCAAT CM :　TCA　CCA　ACCTCCTGT　CCT　CαAττ　Ding GT　CC T GGT TAT CGCTGG ATG TGT CTG CGG CGτ τττ ATCATA TTCCTCTTCATCCTG CTG CTA T GCCTCATCTTCTTA ττGGττ CTT CTG Qλτ TA T CAA GGT ATG Ding τG CCCGTT TGT CCT CTA λττ CCA CGA TCA λCA λQλλ((ACT ACCGGA CCA TCCAAA Enter CCTGCACG ACT CCTGCT CAA GGC input λCTCT ATG TTT CCCTCA TGT TGCTGT ACA AAA CCT ACG GAT GGA AAT TGCACCTG T ATT CCCATCC U TCG TCCTGG GCT TTCGCA AAA TλCCτλ τGGGXG TGG GCCTCA GTCCGT TTCTCT TGG CTCACT TTACTA GTG CG TTT GTT αG TGG ττCGτλ GGG CTT TCCCCCACT GTT TCG CTT TCA CCT ATA TGG ATG ATG TGGτλτ TCG GGC; CCA AGT CTG TACAGCA TCGTG AGT CCCTTT ATA CCG CTG TTA CCA ATT TTCTTT TGT CTCTGGGτ included TAC person τT Sequence number: 28 Sequence type: nucleotide Array length: 106 bl) chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Sl Direct origin: chemical synthesis Sequence number: 29 Sequence type: nucleotide Sequence length: 115 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV S Direct origin: chemical synthesis AC-AAG-AAT-CCT-口＾C-AAT-A(lowGCA-GAG-( C1-3” array number=30 Sequence type: nucleotide Sequence length: 108 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Core Direct origin: chemical synthesis 5-5AT-CTr-+ TA-AAG-GGA-TCC-TCT-11icT -GGG-GGG-AAT-5GA-TGA-CTlow TAG-CTA-CCT -GGG-TGG-GCA-ATA-ATT-TGG-AAG-ATC-CAG -CAT-CTA-(iGG-ACC-+ TG-TAG-TAA-AT low T AG-AC-(Al-3' SEQ ID NO: 31 Sequence type: nucleotide Sequence length: 106 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Core Direct origin: chemical synthesis '5'-5AT-CT Low C%-GAA-TrC-CTG-GGG-CAT-G GA-CAT-TGA-CCC-TTA-TAA-A13A-ATT-TGG- AGC-TAC-TGT-GGA-C5+ + -ACT-CTC-GTT-T TT-GCC-TTC-τ15A-CTT-CTT-TClow TTC-CGT- CAG-CG+-ko.

Sequence number: 32 Sequence type: nucleotide Sequence length: 89 bp chain number knee heavy chain Topology/linear Sequence type Nigetum DNA Origin: HBV Core Direct origin: chemical synthesis 5-GAT-CTC-CTA-GAC-ACC-GCC-TCA-GCT-CT G-TAT-CGA-GAA-r3CC-TTA-GAG-70-CCT-G AG-CAT-TeC-TCA-CCT-CAC-CAT-ACT-GCA-C TC-AGG-CAA-G-CG)-3' Sequence number = 33 Sequence type: nucleotide Sequence length: 25bp chain number knee heavy chain topology bilinear Sequence type Nigetum DNA Origin: HBV Core Direct origin: chemical synthesis 5'-TTG-GAT-CCT-CCA-AC Row Engineering GT-GCC-TTG-( G)-3' sequence number = 34 Sequence type: nucleotide Sequence length: 25 bp chain number knee heavy chain Topology: linear Sequence type Nigetum DNA Origin: HBV Core Direct origin: chemical synthesis 5'-CCT-CTA-GAA-mouth C^-AAT-ATT-CAM-GTA-( C)-Go' SEQ ID NO: 35 Sequence type/nucleotide Sequence length: 588 bp chain number knee heavy chain topology bilinear Sequence type Nigetum DNA Origin: HBV Core Direct origin: PCR amplification TCCAAACCTG TGCCTT GGG TGG CTT TCG GGC CCG QACATT GACCCT τAτ λ input AGAλ Ding Tτ CCA CCTACT GTG GAG ττλ CTCTCG τττ ττG C CT TCT (, Input CTTCτττ CCT TCCGτλ CGA GAT CTCCTA GACACCGCCTCA GCT CTG Ding AT CGA GAA GCCTTA GAG TCTCCT GAG CAT TGCTC A CCT CACCAT ACT GCA CTCAGG CAA GCCλ ττ CTCTGCTGG GGG CAA ττG λDing G input CT CTA GCT λCCTGG GTG GGT input λτ λAτ ττGCAAGλτ CCA GCA TCCAGA GλTCτAGτλ CTCλ input TTλτG ττ　λ入T　ACT　AACλτG　CGT　TTA　AAG　λτCAGG C input Cτλ ττG TGG τDing T CAT ATA TCT TGCC TT ACT τττGGA AGA GAG ACT Gτλ CTT CA A τλT ττGGτCTCττTCCGA GTG TGG ATT CG CACT CCT CCA GCCτ nested AGA CCA CCA)-1T GCCCCT kTG TTA TCA AGA CTTCCG GAA AC T ACT GTT GTT AGA CCA CGG CACCGAGGCA GG TCCCCT AGA AGA AGA ACT CCCTCG CCT CGCAGA CGT AGA TCT CAA TCG CCG CGT C GCAGAAGA TCT CAA TCT CGG GAA TCT CAA TGT τAGFig, IV liver enzyme measurement Fig, Vll Results of chimpanzee experiment 3 Fig, IX Antigen measurement Patient #1 Fig, X Antibody measurement Patient #1 Fig, XI Antigen measurement Patient #2 Fig, Xll antibody measurement Patient #2 Submission of translation of written amendment (Article 184-8 of the Patent Act) June 1993 tr date

Claims (22)

[Claims] 1. a) at least one polypeptide that mediates antigenicity of one or more epitopes; Petide array and b) a carrier capable of presenting epitope sequence a), said polypeptide bonding the de-sequence a) to the support b) by adsorption, arbitrary chemical bonds or sub-valences; combination with a carrier that can be used. 2. Polypeptide sequence a) directly or indirectly represents a T cell activation epitope The combination according to claim 1, characterized in that it mediates the antigenicity of. 3. that the polypeptide sequence a) is a hepatitis B virus polypeptide; Combination according to claim 1 or 2, characterized in that: 4. The polypeptide sequence a) is an HB virus peptide pre-S1, pre-S2, S or the amino acid sequence of one or more members selected from the group consisting of Combination according to any one of claims 1 to 3, characterized in that: 5. The polypeptide sequence a) is i) any conserved epitope comprising at least 6 contiguous amino acid residues; ii) one or several amino acid substitutions; by or iii) an insertion at the N-terminus, at the C-terminus or into the polypeptide sequence a); A claim characterized in that it can be modified by having additional amino acids as The combination according to any one of claims 1 to 4. 6. Claims 1 to 3, characterized in that the polypeptide sequence a) is myristylated. 5. The combination described in any one of 5. 7. The carrier b) is a polysaccharide, a hydrophobic polymer or an inorganic molecule with particulate form. Combination according to any one of claims 1 to 6, characterized in that: 8. Claims 1 to 6, characterized in that carrier b) is a second polypeptide sequence. The combination described in any one of the above. 9. polypeptide sequence b) produces particles having a diameter of at least 10 nm upon secretion; 9. The combination according to claim 8, characterized in that it forms. 10. Polypeptide sequence b) is an HBVS peptide, HBV core antigen, HAV core antigen, surface antigens and HIV core antigens and surface antibodies of poliovirus HAV or HIV. a substantial portion or complete amino acid sequence of a polypeptide selected from the group consisting of Combination according to claim 8 or 9, characterized in that: 11. The polypeptide sequence b) is i) by having any deletion that preserves the ability to form particles; ii) by having one or more by having substitutions of several amino acids, or iii) as an insertion at the N-terminus, C-terminus or into the polypeptide sequence b) Claim 8, characterized in that it can be modified by having additional amino acids. The combination according to any one of -10. 12. Claim 8 characterized in that the polypeptide sequence b) is myristylated. The combination according to any one of -11. 13. Claims: polypeptide sequences a) and b) are linked by a disulfide bond The combination according to any one of items 1 to 6 or 8 to 12. 14. The polypeptide sequences a) and b) are ``hydrophobically fixed'' (mediated by myristic acid). according to any one of claims 1 to 6 or 8 to 13, combined by A combination of 15. Polypeptide sequences a) and b) are joined by a peptide bond, where Optionally a spacer sequence between polypeptide sequence a) and polypeptide sequence b) is inserted, and the spacer sequence is linked to the polypeptide sequence a) via a peptide bond. and b) according to any one of claims 1 to 6 and 8 to 14, characterized in that The combination described in paragraph 1. 16. Any of claims 1 to 15 for the manufacture of a medicament for treating chronic viral hepatitis. Use of the combination described in item (1) above. 17. Recombinant DNA encoding the combination according to any one of claims 1 to 16 molecule comprising at least one first DNA sequence, optionally a second, third and and/or a fourth DNA sequence, wherein i) said at least one first DNA sequence according to any one of claims 1 to 6. encoding at least one polypeptide sequence a) as described; ii) the second DNA sequence is a polypeptide according to any one of claims 8 to 12; iii) said third DNA sequence according to claim 15; code the spacer array for, and iv) said fourth DNA sequence encodes a selectable marker, and said DNA sequence are regulated by DNA elements essential for expression and optionally share a common reading frame. A recombinant DNA molecule characterized by: 18. characterized in that up to 30% of the nucleotides in the first DNA sequence can be substituted; 18. The recombinant DNA molecule according to claim 17. 19. Transcribed by the recombinant DNA according to any one of claims 17 or 18. infected host cells. 20. The host cell is selected from the group consisting of mammalian, yeast and bacterial cells. 20. The host cell according to claim 19, characterized in that: 21. 16. A method for treating chronic viral hepatitis, according to any one of claims 1 to 15. A method comprising administering to a patient the combination described in . 22. 22, wherein said administration is by intravenous or intramuscular injection. Method.